Analysis of Conventional and Unconventional Trafficking of CFTR and Other Membrane Proteins
The cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic transmembrane protein that functions as a cAMP-activated anion channel at the apical membrane of epithelial cells. Mutations in CFTR cause cystic fibrosis and are also associated with monosymptomatic diseases in the lung, pancreas, intestines, and vas deferens. Many disease-causing CFTR mutations, including the deletion of a single phenylalanine residue at position 508 (ΔF508-CFTR), result in protein misfolding and trafficking defects. Therefore, intracellular trafficking of wild-type and mutant CFTR has been studied extensively, and results from these studies significantly contribute to our general understanding of mechanisms involved in the cell-surface trafficking of membrane proteins. CFTR is a glycoprotein that undergoes complex N-glycosylation as it passes through Golgi-mediated conventional exocytosis. Interestingly, results from recent studies revealed that CFTR and other membrane proteins can reach the plasma membrane via an unconventional alternative route that bypasses Golgi in specific cellular conditions. Here, we describe methods that have been used to investigate the conventional and unconventional surface trafficking of CFTR. With appropriate modifications, the protocols described in this chapter can also be applied to studies investigating the intracellular trafficking of other plasma membrane proteins.
Key wordsMembrane protein Unconventional trafficking Cystic fibrosis transmembrane conductance regulator (CFTR) Surface biotinylation Immunofluorescence staining
We thank Shin Hye Noh and Joonhee Park for editorial assistance. This work was supported by grants 2013R1A3A2042197 and 2007-0056092 from the National Research Foundation, the Ministry of Science, ICT & Future Planning, and grant HI14C0070 from the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. The authors have no conflict of interest to disclose.
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